CN108641984B - Urea bacillus SC-7 and application thereof in compost - Google Patents
Urea bacillus SC-7 and application thereof in compost Download PDFInfo
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- CN108641984B CN108641984B CN201810481751.8A CN201810481751A CN108641984B CN 108641984 B CN108641984 B CN 108641984B CN 201810481751 A CN201810481751 A CN 201810481751A CN 108641984 B CN108641984 B CN 108641984B
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Abstract
The invention discloses a strain of Bacillus urealyticum SC-7 and application thereof in compost. The invention provides a strain of Bacillus ureafaciens SC-7 with the preservation number of CGMCC No.14961, which can rapidly grow and reproduce at high temperature. The liquid or solid microbial inoculum containing the bacillus urealyticum is prepared by fermenting and culturing the bacillus urealyticum, and is added into compost, so that the temperature rise time of the compost can be obviously shortened, the high-temperature period of the compost is obviously prolonged, the decomposition of the compost is accelerated, and the degradation of residual antibiotics is promoted. The urea bacillus or the microbial inoculum containing the same has higher removal efficiency on various common antibiotics with different properties and types, thereby effectively removing residual antibiotics and reducing the influence of antibiotic residues on human health and environment.
Description
Technical Field
The invention relates to the technical field of environment and agricultural biology, in particular to a bacillus ureophilus SC-7 and application thereof in compost preparation.
Background
In order to meet the increasing demands of consumers on livestock and poultry products, the breeding mode of China is shifted from scattered household breeding to large-scale intensive breeding. Antibiotics have been widely used for a long time in the intensive livestock and poultry breeding industry because of their excellent effects in promoting growth, preventing and treating diseases in animals. According to statistics, the total usage amount of antibiotics in China in 2013 is about 16.2 ten thousand tons, and 52 percent of the antibiotics are veterinary antibiotics. Most of the antibiotics for livestock and poultry breeding cannot be completely absorbed by animals, about 60-90% of the antibiotics are discharged from animal excrement and urine to the environment in the form of original drugs or primary metabolites, so that a large amount of antibiotics are left, and if the antibiotics are not subjected to effective harmless treatment, the antibiotic resistance genes in the environment can be further proliferated, so that the public health is further threatened.
The high-temperature aerobic composting is one of the effective harmless and recycling treatment means of the livestock and poultry breeding excrement, researches show that the composting is also an effective method for promoting the degradation of residual antibiotics in the breeding excrement, and the antibiotics in the excrement are degraded at high temperature by the heat generated in the aerobic fermentation process of microorganisms. However, the existing composting process usually focuses on fertilizer indexes such as organic matters, total nutrients, water content and the like, and does not pay attention to the degradation of antibiotic residues, so that the commercial organic fertilizer still has antibiotic residues to a certain degree.
The existing technology for removing antibiotics in the composting process of livestock and poultry manure mainly relates to the addition of a conditioner (such as bamboo charcoal, activated carbon, wood chips and attapulgite) and a pretreating agent (acid or alkali), and improves the treatment effect of the composting reaction on the antibiotics by adjusting the water content and the porosity of a pile body by adopting a physical or chemical method. The method for adding the specific conditioner from an external source is influenced by regions or markets and has certain limitations. Currently, only a few studies have been directed to improving the degradation of antibiotics by adding strains of microorganisms (e.g., Phanerochaete chrysosporium). However, most of the strains used by the microbial inoculum in the compost at present are mesophilic strains, such as bacillus, lactobacillus, trichoderma and the like, and although some microorganisms can tolerate the high temperature of the compost, the microorganisms cannot keep the activity and play a role in the high temperature state.
Aiming at the phenomena of antibiotic residue and incomplete degradation, a composting technology capable of rapidly increasing the composting temperature and prolonging the high-temperature time as far as possible is needed to ensure the effective degradation of the antibiotic residue in the excrement and reduce the harm to the environment and human health.
Disclosure of Invention
In order to solve the technical problems in the prior art, the invention aims to provide the urea bacillus SC-7 and the application thereof in composting.
Firstly, the invention provides a strain of Bacillus ureafaciens (Ureibacillus sp.) SC-7. The urea bacillus SC-7 is preserved in China general microbiological culture Collection center (CGMCC for short, the address is No. 3 Xilu No.1 of Beijing city Kogyo, Microbiol research institute of Chinese academy of sciences, zip code 100101) in 24.11 months and 2017, is classified and named as Ureibacillus sp, and the preservation number is CGMCC No. 14961.
The urea bacillus SC-7 is separated from biogas residue high-temperature compost, is a thermophilic bacterium and can grow and reproduce at high temperature. The shape and characteristics of the cells are as follows: bacilli (fig. 1), gram negative, flagellated, runny, form white morphologically irregular colonies. Can grow at 35-60 deg.c, is positive to catalase and oxidase and does not grow under anaerobic condition. The available carbon source includes glucose, arabinose, xylose, mannitol and other monosaccharides; can hydrolyze starch, gelatin, casein and urea.
Further, the invention provides a microbial inoculum containing the Bacillus urealyticum SC-7, wherein the microbial inoculum is a liquid microbial inoculum or a solid microbial inoculum.
Furthermore, the invention provides a preparation method of the microbial inoculum.
The preparation method of the liquid microbial inoculum comprises the steps of carrying out fermentation culture on the urea bacillus SC-7 to obtain zymocyte liquid or treated zymocyte liquid;
the preparation method of the solid microbial inoculum comprises the steps of mixing the zymocyte liquid with a drying agent, and then carrying out spray drying to obtain the solid microbial inoculum. The drying agent includes, but is not limited to, anhydrous calcium salts, preferably anhydrous calcium carbonate or anhydrous calcium chloride.
It should be understood by those skilled in the art that although the microbial inoculum provided by the present invention is prepared by drying a zymogen solution or a zymogen solution, those skilled in the art may also prepare the microbial inoculum by performing other pretreatment on the zymogen solution according to actual needs. And further, other auxiliary material components can be added on the basis of the obtained microbial inoculum to prepare other microbial preparations (such as microbial compost promoters and the like).
Specifically, the preparation method of the liquid microbial inoculum comprises the following steps:
(1) activating strains: under the aseptic condition, selecting the Bacillus urealyticus SC-7 strain stored in a slant culture medium at a low temperature of 4 ℃, streaking on a solid culture medium, and culturing at 50 +/-2 ℃ for 12-24 hours.
(2) Seed culture, namely selecting the culture obtained in the one-ring step (1) to inoculate the culture into a liquid seed culture medium, and carrying out shaking culture on a table for 12-24 hours at the temperature of 45-50 ℃ until the bacterial count reaches 1 × 1031/ml-1 × 107In the order of magnitude of one/ml, obtaining seed liquid.
(3) And (3) fermentation culture, namely inoculating the seed liquid obtained in the step (2) into a fermentation culture medium according to the volume ratio of 1-10%, and performing fermentation culture at the temperature of 45-50 ℃ until the total bacteria number reaches 1 × 1041/ml-1 × 1012Stopping fermentation in the order of magnitude of one/ml, and obtaining a zymophyte liquid which is the liquidA somatic bacterium agent.
And (3) mixing the zymocyte liquid obtained in the step (3) with sterilized anhydrous calcium carbonate, and then carrying out spray drying to obtain the solid microbial inoculum.
In a specific embodiment of the invention, the solid medium is a beef extract peptone solid medium, namely 3.0g of beef extract, 10.0g of peptone, 5.0g of NaCl and 15-20g of agar, the solid medium is supplemented with distilled water to 1L, and the pH value is adjusted to 7.4-7.6 by 1N hydrochloric acid, the seed medium is a beef extract peptone liquid medium, namely 3.0g of beef extract, 10.0g of peptone and 10.0g of NaCl5.0g of distilled water to 1L, and the pH value is adjusted to 7.4-7.6 by 1N hydrochloric acid, and the fermentation medium is prepared from 5g of glucose, 15g of starch, 30g of soybean meal, 1g of corn steep liquor, 2g of disodium hydrogen phosphate and 1g of magnesium sulfate, and the balance is adjusted to 1L by distilled water.
It will be appreciated by those skilled in the art that the purpose of the fermentation is to provide a Bacillus urealyticum SC-7 thallus. The components and amounts of the culture medium and the culture conditions (temperature, rotation speed, etc.) can be adjusted according to the requirements of different growth rates and final bacterial body amounts, so that the selection of the culture medium and the culture conditions for culturing the Bacillus urealyticum SC-7 strain is not limited to the culture medium and the culture conditions provided in the specific embodiment of the invention.
The urea bacillus SC-7 provided by the invention can be rapidly proliferated under a high-temperature condition, so that the temperature rise time of compost is shortened, the time of the high-temperature period of the compost is prolonged, and the composting efficiency is improved. Meanwhile, the degradation of antibiotics in the organic waste can be promoted by utilizing the high-temperature growth characteristic of the urea bacillus SC-7, so that the antibiotic residues are effectively removed.
Therefore, in another aspect, the invention also provides the application of the Bacillus urealyticum SC-7 or the microbial inoculum in purifying organic waste materials.
And the application of the urea bacillus SC-7 or the microbial inoculum in composting. The application is in the compost degradation of antibiotics, the compost decomposition acceleration, the compost temperature rise time shortening and/or the compost high-temperature period prolonging.
The antibiotic may be tetracycline, sulfanilamide, quinolone or macrolide antibiotic, and specifically oxytetracycline, sulfamethoxazole, ciprofloxacin or erythromycin.
It will be appreciated by those skilled in the art that the composting material may contain different organic materials and in particular embodiments of the invention the composting material is predominantly livestock manure, but the choice of composting material may not be limited thereto.
The application is that the Bacillus urealyticum SC-7 or the microbial inoculum is added to the organic waste or the compost. Specifically, the application is to add the microbial inoculum in an amount which is more than or equal to 0.5 per thousand of the mass ratio of the microbial inoculum to the organic waste or the compost.
Preferably, the application method of the liquid microbial inoculum specifically comprises the step of spraying the microbial inoculum to the surface of the compost according to 0.5-3.0 per mill (V/W) of the compost raw materials, so that the bacterial liquid uniformly covers the surface of the compost, and can be sprayed before the compost raw materials are turned over, or sprayed together with a turning machine, and the temperature rise of the compost is promoted.
The application method of the solid microbial inoculum is that the solid microbial inoculum is uniformly spread on the surface of composting raw materials according to 0.5-3.0 per mill (W/W) of the composting raw materials.
The microbial inoculum or the microorganism provided by the invention is divided into a liquid form and a solid form, the effective period of the microbial inoculum in the liquid form is about two months, and the effective period of the microbial inoculum in the solid form is more than six months at normal temperature. The skilled person can select and use the method according to the needs of the actual situation.
The invention has the beneficial effects that:
1. the urea bacillus and the microbial inoculum thereof provided by the invention can utilize macromolecular substances such as starch, protein and the like in organic waste materials to rapidly grow and propagate, so that the time for reaching the highest temperature in a compost pile body is shortened by 1 day, the highest temperature can reach 68.3 ℃, and meanwhile, the time for maintaining the temperature in the pile body at more than 50 ℃ is prolonged by 5 days, thereby realizing rapid temperature rise and longer high-temperature period, promoting compost maturity and improving compost efficiency.
2. The urea bacillus and the microbial inoculum thereof provided by the invention can be effectively proliferated in a high-temperature period of compost, so that the high-temperature period is prolonged, and the effective degradation of residual antibiotics in organic waste is realized.
3. Compared with the selectivity of the microbial inoculum used in the prior art on the effect of antibiotics, the urea bacillus and the microbial inoculum thereof provided by the invention have the removal efficiency of more than 99% on various common antibiotics with different properties and types.
4. The urea bacillus provided by the invention can be rapidly propagated at high temperature, has a shorter fermentation period compared with mesophilic bacteria, and saves the fermentation and cooling costs due to the higher fermentation temperature, thereby saving the production cost of the microbial inoculum.
5. The preparation process and the use method of the microbial inoculum provided by the invention are simple and easy to operate, and can be practically used for composting.
Drawings
FIG. 1 is a diagram showing the form of Bacillus urealyticum SC-7 cells under an optical microscope.
FIG. 2 shows the temperature change of the Bacillus urealyticum SC-7 in the compost of chicken manure.
FIG. 3 shows the concentration change of the antibiotics (oxytetracycline, sulfamethoxazole, ciprofloxacin and erythromycin, respectively) of Bacillus urealyticum SC-7 in chicken manure compost.
Detailed Description
Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Example 1 preparation of liquid inoculum
The liquid microbial inoculum containing the urea bacillus SC-7 is prepared by the following method.
(1) Activating strains: under aseptic conditions, selecting a Bacillus urealyticus SC-7 strain stored in a slant culture medium at a low temperature of 4 ℃, streaking on a fresh beef extract peptone solid culture medium, and culturing for 24 hours at a temperature of 50 +/-2 ℃.
Beef extract peptone medium, beef extract 3.0g, peptone 10.0g, NaCl5.0g, agar 15-20g, adding distilled water to 1L, adjusting pH to 7.4-7.6 with 1N hydrochloric acid, sterilizing at 121 deg.C for 20 min.
(2) Seed culture: preparing a beef extract peptone liquid culture medium: adding water to 0.6g of beef extract, 2g of peptone and 1g of NaCl, supplementing to 200ml of water, adjusting the pH to 7.4-7.6 by using 1N hydrochloric acid, subpackaging into 2 triangular bottles of 250ml, each bottle is 100ml, sterilizing at 121 ℃ for 20 minutes. Inoculating the activated urea bacillus SC-7 in the step (1) into the beef extract peptone liquid culture medium, culturing at 45-50 ℃ at 150-200rpm for 12 hours in a shaking table, and taking the obtained bacterial liquid as seed liquid.
(3) And (2) fermentation culture, namely adding 5L water into a 7L small fermentation tank, adding 25g of glucose, 75g of starch, 150g of soybean meal, 5g of corn steep liquor, 10g of disodium hydrogen phosphate, 5g of magnesium sulfate, 115 ℃, sterilizing for 20min, mixing and inoculating 2 bottles of seed liquid into the fermentation tank when the temperature of a culture medium is reduced to 50 ℃, adding 2.5ml of foam killer serving as an antifoaming agent at the temperature of 50 ℃, sampling once every two hours at the stirring speed of 150rpm, measuring the absorbance at 600nm after dilution, stopping fermentation when the total absorbance value reaches 20, wherein the fermentation liquid is the liquid microbial inoculum.
Example 2 preparation of solid microbial inoculum
The preparation method of the solid microbial inoculum containing the urea bacillus SC-7 is as follows.
(1) Activating strains: under aseptic conditions, selecting a Bacillus urealyticus SC-7 strain stored in a slant culture medium at a low temperature of 4 ℃, streaking on a fresh beef extract peptone solid culture medium, and culturing for 24 hours at a temperature of 50 +/-2 ℃. The preparation of beef extract peptone solid medium was the same as in step (1) of example 1.
(2) And (2) seed culture, namely preparing a beef extract peptone liquid culture medium, namely 4.8g of beef extract, 16g of peptone and 8g of NaCl, adding water to supplement the water to 1.6L, adjusting the pH value to 7.4-7.6 by using 1N hydrochloric acid, subpackaging the mixture into 500ml triangular bottles, filling 160ml of liquid in each bottle, and sterilizing the bottles at 121 ℃ for 20 minutes at 10 ℃ in total, inoculating the activated urea bacillus SC-7 in the step (1) into the beef extract peptone liquid culture medium, rotating the bottles at 50 ℃ for 200 r/min, and performing shake culture for 12 hours to obtain a bacterial liquid as a seed liquid.
(3) And (2) fermentation culture, namely adding 75L of water into a 100L fermentation tank, adding 375g of glucose, 1125g of starch, 30g of soybean meal, 2250g of corn steep liquor, 150g of disodium hydrogen phosphate, 75g of magnesium sulfate, sterilizing at 121 ℃ for 20 minutes, cooling the temperature of a culture medium to 50 ℃, mixing 10 bottles of seed liquor into 3 bottles, inoculating the 3 bottles of seed liquor into the fermentation tank, fermenting at the temperature of 50 ℃, under the pressure of 0.05Mpa, under the ventilation of 80L/minute and at the stirring speed of 150rpm, adding 40ml of foam killer serving as a defoaming agent, sampling once every two hours, measuring the absorbance at the wavelength of 600nm after dilution until the absorbance value of the fermentation liquor reaches 2.0, and stopping fermentation.
(4) And (4) mixing the bacterial liquid obtained in the step (3) with sterilized anhydrous calcium carbonate, and performing spray drying to obtain the solid microbial inoculum.
Example 3 application of Bacillus urealyticum SC-7 microbial inoculum in compost
Experimental materials: the experimental chicken manure is taken from a certain chicken farm in Mitsui of Beijing, 5 days before the manure is collected, the poultry applies feed without antibiotics, and fresh manure collected on the 6 th day is naturally dried for composting.
The experimental scheme is as follows: an experimental group (with bacteria) and a control group (without bacteria) are set, each group is repeated, the main compost components are 9.2kg of mushroom residues, 18.3kg of chicken manure and 60% of water content, and oxytetracycline, sulfamethoxazole, ciprofloxacin and erythromycin are artificially added into the compost to enable the final concentration of each antibiotic to reach 60 mg/kg. Experimental groups: 30g of high-temperature urea bacillus SC-7 bacterial liquid is taken, diluted by 300g of clear water and uniformly mixed with compost materials. And (4) periodically detecting the internal temperature of the compost, sampling and detecting the concentration of the antibiotics in the compost.
And detecting the concentration of the antibiotic by adopting a solid-phase extraction combined with an ultra-high performance liquid chromatography-tandem mass spectrometry method.
The experimental results are as follows: the results of livestock and poultry manure composting experiments show that the temperature in the pile of the experimental group added with the urea bacillus fungicide is rapidly increased (figure 2), the highest temperature can be reached by 68.3 ℃ in 2 days, compared with a control group, the time for reaching the highest temperature is advanced by 1 day, the fermentation delay period is basically avoided, and the temperature in the pile of the experimental group added with the urea bacillus fungicide is increased by 2.9 ℃ compared with the control group; meanwhile, the temperature in the pile of the experimental group is maintained for 20 days at more than 50 ℃, and is prolonged by about 5 days compared with the control group; after the high-temperature period is finished, the removal rate of the oxytetracycline, the sulfamethoxazole, the ciprofloxacin and the erythromycin reaches over 99 percent (figure 3), and compared with the removal rate of a control group of 80-90 percent, the removal rate of the antibiotics of the experimental group added with the microbial inoculum containing the urea bacillus is obviously improved, which indicates that the rapid degradation of the antibiotics in the livestock and poultry manure can be promoted by adding the microbial inoculum.
In conclusion, the urea bacillus SC-7 or the microbial inoculum containing the urea bacillus SC-7 provided by the invention can obviously shorten the temperature rise time of livestock and poultry raising manure compost, prolong the high-temperature period time and effectively remove various antibiotics remained in the livestock and poultry manure.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the technical principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A strain of urea bacillus (Ureibacillus sp.) SC-7 is characterized in that the strain is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No. 14961.
2. A microbial inoculum comprising Bacillus urealyticum (SC-7) as defined in claim 1, wherein the microbial inoculum is a liquid microbial inoculum or a solid microbial inoculum.
3. The method for preparing the microbial inoculum according to claim 2, wherein the bacillus ureabalis (Ureibacillus sp.) SC-7 according to claim 1 is subjected to fermentation culture to obtain zymocyte liquid, namely a liquid microbial inoculum; and mixing the zymophyte liquid with a drying agent, and then carrying out spray drying to obtain the solid microbial inoculum.
4. The method of claim 3, wherein the drying agent is an anhydrous calcium salt.
5. The method of claim 4, wherein the drying agent is anhydrous calcium carbonate or anhydrous calcium chloride.
6. A Bacillus urealyticum (Ureibacillus sp.) SC-7 as defined in claim 1 or a microbial inoculum as defined in claim 2 for use in composting antibiotics selected from one or more of oxytetracycline, sulfamethoxazole, ciprofloxacin and erythromycin.
7. Use of a Bacillus urealyticum (Ureibacillus sp.) SC-7 according to claim 1 or a microbial inoculum according to claim 2 for accelerating compost maturity.
8. Use of a bacillus ureabarius sp-7 according to claim 1 or a bacterial agent according to claim 2 for shortening the temperature rise time and/or prolonging the high temperature period of compost.
9. The use according to any one of claims 6 to 8, characterized in that the Bacillus ureafaciens (Ureibacillus sp.) SC-7 or a microbial inoculum containing it is added to compost.
10. The use as claimed in claim 9, wherein the microbial inoculum is added in an amount of 0.5% o by mass or more of the compost.
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